1. Field of the Invention
The present invention relates to analysis of data of nucleic acid amplification reactions. More specifically, the invention relates to an information system and method for making determinations regarding chemical and/or biological reactions. The invention also involves an alternate method of quantifying nucleic acids in a sample comprising amplification of a target nucleic acid and analysis of data obtained during the amplification reaction. The invention further involves a diagnostic system and/or kit using real-time nucleic acid amplification including, but not limited to, PCR analysis.
2. Discussion of the Art
In many different industrial, medical, biological, and/or research fields, it is desirable to determine the quantity of a nucleic acid of interest. Some methods of quantifying nucleic acids of interest involve amplifying them and observing a signal proportional to the quantity of amplified products made; other methods involve generating a signal in response to the presence of a target nucleic acid, which signal accumulates over the duration of the amplification reaction. As used herein, nucleic acid amplification reaction refers both to amplification of a portion of the sequence of a target nucleic acid and to amplification and accumulation of a signal indicative of the presence of a target nucleic acid, with the former often being preferred to the latter. The quantification of nucleic acids is made more difficult or less accurate or both because data captured during amplification reactions are often significantly obscured by signals that are not generated in response to the target nucleic acid (i.e., noise). Furthermore, the data captured by many monitoring methods can be subject to variations and lack of reproducibility due to conditions that can change during a reaction or change between different instances of a reaction. In view of the above, there is a need to develop improved means of quantifying a nucleic acid. Where quantification of nucleic acids is enabled by amplification reactions, there is also a need to improve current methods of detecting suspect or invalid amplification reactions. There further remains a need to improve current abilities to distinguish between amplification reactions that do not detect a target nucleic acid (i.e., negative reactions) from weak signals obtained from amplification reactions suffering from low quantities of a target nucleic acid in a sample, a degree of inhibition of the amplification reaction, or other causes. The present invention provides improvements in these areas as is disclosed below.
A non-exhaustive list of references providing background information regarding the present invention follows:    Livak, K. and Schmittgen, T., Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 22DDCT Method, METHODS 25: 402-408 (2001) doi:10.1006/meth.2001.1262.    Bustin S A, Absolute quantification of mRNA using real-time reverse transcription PCR assays, Journal of Molecular Endocrinology 25: 169-193 (2000).    Bustin S A., Quantification of mRNA using real-time reverse transcription PCR: trends and problems, J Mol Endocrinol. 29: 23-29 (2002).While the inventors cannot guarantee that the following website will remain available and do not necessarily endorse any opinions expressed therein, an interested person may wish to refer to the world wide web at wzw.tum.de/gene-quantification/index.shtml for useful background information.
The discussion of any works, publications, sales, or activity anywhere in this submission, including in any documents submitted with this application, is not intended to be an admission of any manner that any such work constitutes prior art, unless explicitly stated to the contrary. Similarly, the discussion of any activity, work, or publication herein is not an admission that such activity, work, or publication was known in any particular jurisdiction.
Real-time PCR is an amplification reaction used for the quantification of target nucleic acids in a test sample. Conventionally, skilled artisans typically view the amplification reaction as comprising three distinct phases. First, there is a background or baseline phase, in which the target nucleic acid is being amplified but the signal proportional to the quantity of the target nucleic acid cannot be detected because it is too small to be observed relative to signals independent of the target (sometimes called “background” or “background signal”). Next, there is a logarithmic phase in which the signal grows substantially logarithmically because the signal is substantially proportional to the quantity of target nucleic acid in the amplification reaction and is greater than the background signal. Finally, the growth in the signal slows during a “plateau” phase reflecting less than logarithmic amplification of the target nucleic acid. As is known in the art, the time at which the logarithmic phase crosses a threshold value, which is a value somewhat greater than the value of the background signal, is reproducibly related to the log of the concentration of the target nucleic acid. This prior art method is generically referred to as the Ct method, perhaps so named for the Cycle at which the signal crosses the threshold. Ct analysis is reasonably reproducible and accurate, but suffers from some drawbacks, which need not be discussed here to understand the present invention.
U.S. Pat. No. 6,303,305 discloses a method of quantification of nucleic acids employing PCR reactions. The method disclosed employs the nth derivative of the growth curve of a fluorescent nucleic acid amplification reaction. This method effectively avoids the need to perform a baseline correction, but provides no reliable method of determining reactive from non-reactive samples, and does not reasonably suggest how to use an nth derivative calculation to assess the validity of the results obtained. In addition, nucleic acid amplification signals resulting from any artifacts in the system (e.g., crosstalk or positive bleedover—defined infra) cannot be distinguished from true positive responses using the methods disclosed therein and can lead to false positive results. However, the first derivative calculation disclosed by U.S. Pat. No. 6,303,305 provides an efficiency related value that is useful in the context of the present invention. The skilled artisan can refer to U.S. Pat. No. 6,303,305 for additional details relating to calculation of a first derivative of a nucleic acid amplification signal growth curve. U.S. Pat. No. 6,303,305 is incorporated by reference only in the United States of America, and other jurisdictions permitting incorporation by reference, to the extent it discloses the calculation of the first derivative of a nucleic acid amplification growth curve. However, U.S. Pat. No. 6,303,305 does not disclose or suggest the uses of this efficiency related value described in this disclosure (below).
Co-owned U.S. Provisional Patent Application No. 60/527,389, filed Dec. 6, 2003, discloses a method for analyzing a nucleic acid amplification reaction in which the log of the signal from an amplification reaction is examined for the maximum gradient or slope. This value, which for any data set corresponds to a point a certain period of time or number of cycles after the initiation of the amplification reaction, is called the MGL of the reaction. The MGL is useful in certain embodiments of the present invention, particularly in those that distinguish qualitatively those samples comprising little target nucleic acid from those samples that do not contain target nucleic acid. U.S. Patent Application No. 60/527,389, filed Dec. 6, 2003 is incorporated herein by reference in its entirety.